Description (adapted from the application). The lack of an adequate cell culture system to study HCV replication has been a major obstacle to progress in understanding the pathogenesis of hepatitis C. The goal of this laboratory has been to establish cultures of non-transformed human hepatocyte, which retain the expression of liver-specific markers and are suitable for the study of interactions between HCV and its natural host, the normal human hepatocyte. This project describes the general features of a newly established culture system using cultured human fetal hepatocytes (HFH). Cells transfected with full length HCV RNA (WT) but not with a 3'delta-deletion mutant, express core protein and show virus replication as demonstrated by strand-specific in situ hybridization (ISH). In cultures transfected with WT HCV RNA but not in those transfected with mutant RNA, cells are vacuolated, grow in a nodular pattern and apoptosis demonstrated by TUNEL or 'live dead' assays is evident. It is proposed that apoptotic elimination of sensitive cells, preservation of permissive cells in which the virus persists and activation of cytokine mediated viral clearance mechanisms are events that occur during acute HCV infection and set the stage for the outcome of the infection. Experiments have been designed to test these hypotheses and to determine in clinical samples whether specific mutations and quasispecies evolution may correlate with HCV replication and cytopathic effects in cultured human hepatocytes. Experiments described in Aim 1 will determine the main parameters of viral replication and infectivity in HFH cultures transfected with HCV RNA or infected with patient sera and explore new methods to increase the efficiency of transfection or infection. Aim 2 will examine the expression of apoptotic markers in HFH cultures and investigate whether oxidant damage, Fas-Fas-L expression and cytokine-mediated signaling pathways are important mechanisms in HCV-induced hepatocyte apoptosis. Project 1 is directly linked to Projects 2 and 3 and Core A. The cDNA microarray gene expression analysis described in Project 2 will provide a comprehensive picture of the HFH culture system, HCV cytopathic effects and IFN signaling. Clinical samples obtained in studies described in Project 3 and prepared by Core A will be used to test the hypothesis that clinical isolates well characterized as to genotype, quasispecies and disease outcome in patients will differ in their capacity to produce cytopathic effects in hepatocytes.